This proposal focuses on the effects of estradiol and its metabolites on the function and expression of Mrp2 (ABCC2), the ABC transporter that mediates the biliary excretion of glucuronide and glutathione conjugates from the hepatocyte into bile. The proposal builds on our findings that 1) Mrp2-mediated transport of estradiol-17Beta-(Beta-D-glucuronide) (E217G) is essential for its cholestatic activity, 2) E217G causes endocytic retrieval of Mrp2 from the canalicular membrane that coincides with decreased bile flow, and 3) expression of Mrp2 protein, but not Mrp2 mRNA, is decreased in pregnancy. Aim 1 will test the hypothesis that transport of cholestatic E217G and the choleretic estradiol-3-glucuronide (E23G) by rat Mrp2 and human MRP2 is mediated by overlapping but non-identical substrate binding sites. We will use an Mrp2/MRP2 baculovirus expression system in Sf9 cells and probe the substrate binding sites of E217G vs E23G. We will also determine if women with intrahepatic cholestasis of pregnancy have polymorphisms in MRP2 that alter their transport of E217G vs E23G. Aim 2 will test the hypotheses that A) E217G causes endocytic retrieval of Mrp2 and other transporters critical to bile formation from the canalicular membrane leading to inhibition of flow, and B) agents that protect against cholestasis do so by either inhibiting Mrp2 transport of E217G or prevention of transporter retrieval. We will use confocal immunomicroscopy to monitor endocytic retrieval of transporters from and their exocytic insertion into the canalicular membrane. Aim 3 will characterize the changes in Mrp2 expression in pregnancy and test the hypotheses that A) estrogens mediate the decreased hepatic Mrp2 expression in pregnancy, B) Mrp2 is subject to transcript-specific translational control conferred by regulatory elements in the 5' or 3' untranslated regions of Mrp2 mRNA and C) pregnancy and estrogens increase the degradation of Mpr2 protein. We will use polysomal distribution analysis of Mrp2 mRNA, translation assays in HepG2 cells and determine the degradation half-life of Mrp2 protein in control, pregnant, and estrogen-treated rats. Significance: Characterization of the mechanisms by which estrogens decrease Mrp2/MRP2 function and expression can lead to 1) improved drug therapy for women in pregnancy, 2) development of therapeutic measures to increase MRP2 function in cholestatic liver disease, and 3) methods to screen for cholestatic toxicity in drugs under development.